1. Field of the Invention
The invention relates to a method for obtaining hyperpolymeric hemoglobins.
2. Description of Related Art
Chemical modifications of native hemoglobins for the purpose of varying the oxygen affinity or their degree of polymerization are performed with the aim of developing a synthetic oxygen carrier. This carrier is intended to support oxygen transportation by the blood in man.
Artificial oxygen carriers can be infused, for example, after an accident resulting in bleeding that is difficult to control, or in the case of a risk of infection (hepatitis, AIDS) as a substitute for a temporarily unavailable matching replacement blood. This also applies when a person is in shock due to an insufficiency of blood volume. An artificial oxygen carrier can possibly break through such insufficiency shock easier than banked blood because the preserved erythrocytes stiffen increasingly in storage and therefore have a reduced ability to pass through the capillaries, since they have to be deformed greatly while passing through the capillaries. Such artificial oxygen carriers contain no blood group antigens. Therefore they can be used universally and without cross matching beforehand.
In comparison with banked blood, an artificial oxygen carrier furthermore offers an advantage in cases in which, despite ABO and Rh factor compatibility, there is a danger of an immunological over-reaction against leukocytes. An artificial oxygen carrier, on the other hand, is entirely free of white blood corpuscles which might cause these reactions. Filtration of banked blood, through cotton for example, has proven insufficient to eliminate white blood cells. It was shown many years ago by animal tests that blood volume insufficiency shock can be combated more effectively with artificial oxygen carriers than with simple plasma expanders (review article on this subject: R. Pabst, Med. Klin. 72 (1977), 1555-1562.)
It is furthermore to be expected that chronic circulatory disturbances of a coronary, cerebral or peripheral nature can be treated effectively by means of appropriate polyhemoglobin solutions. Furthermore, oxygen deficiency conditions without circulatory deficiency, such as chronic anemia, can be treated effectively with such solutions. This is shown by the fact that oxygenated hemoglobin can yield its freely dissolved oxygen better than oxygen "packed" in erythrocytes or liposomes (see below). These indications are estimated to form ten times the market potential of "blood make-up" in the form of volume replacement. In such cases the artificial oxygen carrier must be administered as a hypooncotic additive, as blood replacement.
For the preparation of artificial oxygen carriers various approaches have been used, namely,
1. The use of emulsions of fluorinated hydrocarbons in which oxygen is very soluble (for a review: Issues from the Vth International Symposium on Blood Substitutes, Artificial Cells, Blood Substitutes, and Immobilization Biotechnology 22 (1994).) This method, however, has the disadvantage that when fluorinated hydrocarbons are used tissue reactions occur due to macrocytic cells; of decisive importance here is a critical size of the vesicles, which is about 0.3 .mu.m. PA0 2. The microencapsulation of concentrated hemoglobin solutions in phospholipid vesicles to form so-called artificial erythrocytes or "hemosomes" (Review article: Gaber et al., Encapsulation of Hemoglobin in Phospholipid Vesicles; Preparation and Properties of a Red Cell Surrogate in "The Red Cell, Sixth Ann Arbor Conference," G. J. Brewer (publisher); Alan R. PA0 3. The preparation of appropriate hemoglobin solutions, also with covalent bonding of the hemoglobin to dextran, by which renal excretion is said to be reduced; dextrans tend, however, to stimulate the immune system. PA0 4. Crosslinking the hemoglobin molecules with one another likewise prevents renal excretion. In addition, there is an advantage over the dextran-bound hemoglobins of a higher oxygen binding capacity. Recently it has been possible by crosslinking to obtain soluble hyperpolymeric hemoglobins whose colloid osmotic pressure with respect to standard pressure (32 mbar) in the presence of the necessary concentrations is negligible (Potzschke et al., Advances in Experimental Medicine and Biology; N. Back et al. (Eds.) Vol. 345, 205-213, Plenum Press New York 1994). This approach combines most of the advantages referred to.
Liss, Inc., New York, (1984), 179-190).) Here the danger of macrocytic activation is involved. As in par. 1, a change of the artificial emulsifiers into the (dynamic) membranes of the cell is probable, so that the function of these membranes can be disturbed.
The various approaches mentioned are reflected in the following patents:
Patent DE-OS 24 17 619 describes, for example, the synthesis of polymerized, linked hemoglobin as a substitute plasma protein, hemoglobin linked with dicarboxylidat [sic] being prepared.
Patent DE-OS 27 14 252 describes the preparation of hemoglobin linked with pyrodoxal phosphate.
Patent DE-OS 30 29 307 relates to an artificial carrier which is made by covalent linking of a polysaccharide, dextran for example, with cell-free hemoglobin. Patent BE-PS 838 933 describes the preparation of a water-soluble, linked, polymerized hemoglobin by the reaction of free hemoglobin with a polyfunctional linking agent, followed by stopping the reaction with inactivating agents. A polymeric hemoglobin is made with a molecular weight of 61,000 to 1,000,000 daltons.
U.S. Pat. No. 4,001,401 relates to a linked, polymerized hemoglobin as a plasma expander with a molecular weight of 64,000 to 1,000,000 daltons, which is obtained with the linking agents, glutaraldehyde, hexamethylene diisocyanate or butadiene diepoxide.
European Patent EP 0 201 618 describes a method for the preparation of extra-high molecular weight, compact, soluble polymers of hemoglobin from a highly concentrated solution of monomeric hemoglobin. These polymers of high molecular weight are referred to as hyperpolymers.
In Patent DE-PS 37 14 351, this process is simplified in that erythrocytes are used directly, and the crosslinking agent no longer needs to be added in a lipid phase.
In the preparation of suitable modified hemoglobin solutions for use in clinical practice, the necessity arises of keeping the viscosity of the solution as low as possible. The viscosity of the blood co-determines the so-called total peripheral resistance of the organism. If it is too great, the circulatory system will no longer tolerate it. In particular, an additional burden is placed on the heart. In the case of dissolved hyperpolymers and also hemoglobin polymers, such problems are intensified if polymerization to chain molecules occurs.
Einstein's viscosity law says that uniformly large spheres in a fluid have a minimal viscosity regardless of their radius. This viscosity depends solely on the overall volumetric content of the spheres in the solution. For the viscosity of the solutions to remain low, the polymer molecules must accordingly be so compact--say "spherical"--that with the viscosity of the plasma at a minimum, a maximum amount of the oxygen carrier can be transported.
Therefore, to minimize the viscosity it is important to be able to produce oxygen carrying molecules that are as uniform as possible. Such molecules are also found in nature, e.g., in the earthworm. The uniformity of the molecules and the achievement of a very high molecular weight is a requirement of an artificial oxygen carrier which will satisfy the requirement of a negligible colloidal osmotic pressure. To achieve this, at least all of the hyperpolymeric hemoglobin content in question must be removed.
With EP Patent 0 201 618 and German Patent 37 14 351, the problem of linking hemoglobin to form compact, but soluble, giant molecules can be considered solved. The methods described therein, however, lead to a hyperpolymer mixture with a wide distribution of the molecular weight and a severely disproportional increase of the viscosity with increasing concentration (Bernikol and Burkhard, Adv. Exp. Biol. Med. 248 (1989) 335-340), especially in the range of concentration in which the artificial carrier is to be used.